Applications

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application

Abreviews

Notes

WB

Use a concentration of 1 µg/ml. Detects a band of approximately 29 kDa (predicted molecular weight: 29 kDa).

ICC/IF

Use a concentration of 1 µg/ml.

IHC-P

Use at an assay dependent concentration.

IHC-FoFr

Use at an assay dependent concentration. PubMed: 21895533

Flow Cyt

Use 0.05µg for 106 cells.

IP

Use at an assay dependent concentration.

Target

Function

This protein is an auxiliary protein of DNA polymerase delta and is involved in the control of eukaryotic DNA replication by increasing the polymerase's processibility during elongation of the leading strand. Induces a robust stimulatory effect on the 3'-5' exonuclease and 3'-phosphodiesterase, but not apurinic-apyrimidinic (AP) endonuclease, APEX2 activities. Has to be loaded onto DNA in order to be able to stimulate APEX2.

Sequence similarities

Belongs to the PCNA family.

Post-translationalmodifications

Upon methyl methanesulfonate-induced DNA damage, mono-ubiquitinated by the UBE2B-RAD18 complex on Lys-164. This induces non-canonical polyubiquitination on Lys-164 through 'Lys-63' linkage of ubiquitin moieties by the E2 complex UBE2N-UBE2V2 and the E3 ligases, HLTF, RNF8 and SHPRH, which is required for DNA repair. 'Lys-63' polyubiquitination prevents genomic instability on DNA damage. Monoubiquitination at Lys-164 also takes place in undamaged proliferating cells, and is mediated by the DCX(DTL) complex, leading to enhance PCNA-dependent translesion DNA synthesis.Acetylated in response to UV irradiation. Acetylation disrupts interaction with NUDT15 and promotes degradation.

Cellular localization

Nucleus. Forms nuclear foci representing sites of ongoing DNA replication and vary in morphology and number during S phase. Together with APEX2, is redistributed in discrete nuclear foci in presence of oxidative DNA damaging agents.

ab18197 staining PCNA in tissue sections of the goat spleen by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed. Samples were then blocked with 1% B.S.A. for 10 minutes at 21ºC followed by incubation with the primary antibody for 2 hours at 1/4000. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/250 dilution.

ab18197 staining PCNA in tissue sections of the marmoset spleen by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed. Samples were then blocked with 1% B.S.A. for 10 minutes at 21ºC followed by incubation with the primary antibody for 2 hours at 1/6000. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/250 dilution.

ab18197 staining PCNA in tissue sections of the cow spleen by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed. Samples were then blocked with 1% B.S.A. for 10 minutes at 21ºC followed by incubation with the primary antibody for 2 hours at 1/4000. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/250 dilution.

ab18197 staining PCNA in tissue sections of the sheep spleen by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed. Samples were then blocked with 1% B.S.A. for 10 minutes at 21ºC followed by incubation with the primary antibody for 2 hours at 1/6000. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/250 dilution.

ab18197 staining PCNA in tissue sections of the rat brain by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed. Samples were then blocked with 1% B.S.A. for 10 minutes at 21ºC followed by incubation with the primary antibody for 2 hours at 1/10000. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/250 dilution.

ab18197 staining PCNA in tissue sections of the mouse brain by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed. Samples were then blocked with 1% B.S.A. for 10 minutes at 21ºC followed by incubation with the primary antibody for 2 hours at 1/6000. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/250 dilution.

ab18197 staining PCNA in Monkey COS cell pellet by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed. Samples were then blocked with 1% B.S.A. for 10 minutes at 21ºC followed by incubation with the primary antibody for 2 hours at 1/4000. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/250 dilution.

ICC/IF image of ab18197 stained Hela cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab18197, 1µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

ab18197 staining PCNA in Zebrafish gastrula embryos by Immunocytochemistry/ Immunofluorescence (wholemount).Zebrafish embryos were fixed overnight at 4°C when they had reached 60% epiboly. Cells were fixed in formaldehyde, permeabilized using Proteinase K, blocked with 2% goat serum for 2 hours at 20°C and then incubated with ab18197 at a 1/500 dilution for 16 hours at 4°C. The secondary used was an Alexa-Fluor 488 conjugated goat anti-rabbit polyclonal used at a 1/500 dilution. Cells were post-fixed in PFA for 20 minutes at room temperature after extensive washing of the secondary antibody.The left panel shows DAPI stained nuclei, the center panel is PCNA staining, and the right panel is the merged image.

ICC/IF image of ab18197 stained NIH/3T3 cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab18197, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

Panel A does not show the Alexa Fluor® 488 channel, Panel B shows the specfic nuclear staining by ab18197.

ab18197 staining PCNA in SK-N-SH cells treated with KN-62 (ab120421), by ICC/IF. Increase in PCNA nuclear expression correlates with increased concentration of KN-62, as described in literature.The cells were incubated at 37øC for 24h in media containing different concentrations of ab120421 (KN-62) in DMSO, fixed with 100% methanol for 5 minutes at -20øC and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab18197 (1 æg/ml) was performed overnight at 4øC in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.